Project description:X-linked inhibitor of apoptosis (XIAP) is the most potent endogenous caspase inhibitor preventing cell death via caspase-9, -7 and -3 (initiator and executioner caspase pathways). Using short hairpin RNA (shRNA) against XIAP, stably expressed in a parent HCT116 human colon cancer cell line, a series of clones have been developed. XIAP mRNA levels were established by RT-PCR, the four X (XIAP knockdown) clonal cell lines show 82-93% reduction in XIAP mRNA when compared to the four L (luciferase control) cell lines. Immunoblot analysis showed a 67-89% reduction in XIAP protein in X cell lines compared to L. RNA was analysed by microarray and XIAP knockdown was confirmed in 7 probe sets, there was no significant compensation of other IAP family members. XIAP knockdown induced a 2-fold increase in the basal level of apoptosis without modification of caspase 3/7 activity. Finally, XIAP knockdown sensitises cells to radiotherapy by 20%, to recombinant TRAIL by a 3-fold factor, and to paclitaxel and docetaxel by >2 fold factor. Future work should focus on targeted agents such as rhTRAIL in combination with strategies to down regulate XIAP. XIAP antisense is now in clinical development in oncology.
Project description:Eight rounds of selection of cancer cells invading through matrigel-coated chamber was performed to obtain highly invasive colon cancer sublines HCT116-I8 and RKO-I8. Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC) technology was used to identify the differently expressed proteins and the proteomics data was analyzed by ingenuity pathway analysis (IPA). PAK1-PBD immunoprecipitation combined with Western blot were carried out to determine Cdc42 activity, and qRT-PCR and Western blot were used to determine gene expression. The functional role of Cdc42BPA and Cdc42 pathway in colon cancer invasion was studied by loss-of-function experiments including pharmacological blockade, siRNA knockdown, chamber invasion, and WST-1 assays. Human colon cancer tissue microarray was analyzed by immunohistochemistry for overexpression of Cdc42BPA and its correlation with clinicopathological parameters and patient survival outcomes.
Project description:NADPH oxidases (NOXs) are a family of transmembrane proteins that generates reactive oxygen species. We previously reported that patients with colon cancer who had high NOX5 expression had poor prognosis. However, no studies have studied the cellular functions of NOX5 in colon cancer. This study aimed to clarify the relationship between NOX5 and cancer development using an in vitro model. Real-time quantitative PCR was performed to determine the NOX5 expressions levels of colon cancer cell lines. NOX5 knockdown experiments were conducted, and the impact on cell proliferation, migration, and invasion were analyzed. In addition, mRNA microarray was conducted to assess changes in gene profile. NOX5 mRNA expression was high in HCT116 cells and moderate in SW48 cells. NOX5 knockdown significantly inhibited cell migration and invasion in both HCT116 and SW48 cells; however, NOX5 knockdown reduced cell proliferation in only HCT116 cells. mRNA microarrays showed a strong relationship between NOX5 expression levels and integrin-linked kinase signaling pathways. The NOX5 expression in colon cancer cells affects cancer progression, especially cell motility. NOX5 may be a novel therapeutic target for the future development of treatments for colon cancer.